The HIV Prevention Trials Network (HPTN) 052 enrolled serodiscordant couples. HIV-infected index participants reported no prior antiretroviral (ARV) treatment at enrollment. ARV drug testing was performed retrospectively using enrollment samples from a subset of index participants. ARV drugs were detected in 45/96 (46.9%) participants with undetectable viral load (VL), 2/48 (4.2%) participants with low VL, and 1/65 (1.5%) participants with high VL (P<0.0001). ARV drugs were also detected in follow-up samples from participants who were not on study-administered treatment. ARV drug testing may be useful in addition to self-report of ARV drug use in some clinical trial settings.